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infection causes considerable human morbidity and may develop into a deadly visceral form in endemic regions. The parasite infects macrophages where they can replicate intracellularly. Furthermore, they modulate host immune responses by using virulence factors (lipophosphoglycan, glycoprotein-63, and others) that promote survival inside the cells. Extracellular vesicles (EVs) released by parasites are important for cell-cell communication in the proinflammatory milieu modulating the establishment of infection. However, information on the ability of EVs from different species to modulate inflammatory responses is scarce, especially from those species causing different clinical manifestations (visceral vs. cutaneous). The purpose of this study was to compare macrophage activation using EVs from three species from New World including , and . EVs were released from promastigote forms, purified by ultracentrifugation and quantitated by Nanoparticle Tracking Analysis (NTA) prior to murine macrophage exposure. NTA analysis did not show any differences in the EV sizes among the strains. EVs from and failed to induce a pro-inflammatory response. EVs from both WT and LPG-deficient mutant (LPG-KO) did not show any differences in their interaction with macrophages, suggesting that LPG solely was not determinant for activation. On the other hand, EVs from were immunomodulatory inducing NO, TNF-α, IL-6, and IL-10 via TLR4 and TLR2. To determine whether such activation was related to NF-κB p65 translocation, THP-1 macrophage cells were exposed to EVs. In the same way, only EVs from exhibited a highly percentage of cells positive for NF-κB. Our results suggest an important role of EVs in determining the pattern of immune response depending on the parasite species. For , LPG was not determinant for the activation.

Lipophosphoglycan (LPG) is the major surface glycoconjugate having importance during the host-parasite interface. ( ) displays a spectrum of clinical forms including: typical cutaneous leishmaniasis (TL), mucocutaneous (ML), and atypical lesions (AL). Those variations in the immunopathology may be a result of intraspecies polymorphisms in the parasite's virulence factors. In this context, we evaluated the role of LPG of strains originated from patients with different clinical manifestations and the sandfly vector. Six isolates of were used: M2903, RR051 and RR418 (TL), RR410 (AL), M15991 (ML), and M8401 (vector). LPGs were extracted and purified by hydrophobic interaction. Peritoneal macrophages from C57BL/6 and respective knock-outs (TLR2 and TLR-4 ) were primed with IFN-γ and exposed to different LPGs for nitric oxide (NO) and cytokine production (IL-1β, IL-6, IL-12, and TNF-α). LPGs differentially activated the production of NO and cytokines via TLR4. In order to ascertain if such functional variations were related to intraspecies polymorphisms in the LPG, the purified glycoconjugates were subjected to western blot with specific LPG antibodies (CA7AE and LT22). Based on antibody reactivity preliminary variations in the repeat units were detected. To confirm these findings, LPGs were depolymerized for purification of repeat units. After thin layer chromatography, intraspecies polymorphisms were confirmed especially in the type and/size of sugars branching-off the repeat units motif. In conclusion, different isolates of from different clinical forms and hosts possess polymorphisms in their LPGs that functionally affected macrophage responses.

BACKGROUND: Leishmania enriettii is a species non-infectious to man, whose reservoir is the guinea pig Cavia porcellus. Many aspects of the parasite-host interaction in this model are unknown, especially those involving parasite surface molecules. While lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) of Leishmania species from the Old and New World have already been described, glycoconjugates of L. enriettii and their importance are still unknown. METHODS: Mice peritoneal macrophages from C57BL/6 and knock-out (TLR2 −/−, TLR4 −/−) were primed with IFN-γ and stimulated with purified LPG and GIPLs from both species. Nitric oxide and cytokine production were performed. MAPKs (p38 and JNK) and NF-kB activation were evaluated in J774.1 macrophages and CHO cells, respectively. RESULTS: LPGs were extracted, purified and analysed by western-blot, showing that LPG from L88 strain was longer than that of Cobaia strain. LPGs and GIPLs were depolymerised and their sugar content was determined. LPGs from both strains did not present side chains, having the common disaccharide Gal(β1,4)Man(α1)-PO₄. The GIPL from L88 strain presented galactose in its structure, suggestive of type II GIPL. On the other hand, the GIPL of Cobaia strain presented an abundance of glucose, a characteristic not previously observed. Mice peritoneal macrophages from C57BL/6 and knock-outs (TLR2 -/- and TLR4 -/-) were primed with IFN-γ and stimulated with glycoconjugates and live parasites. No activation of NO or cytokines was observed with live parasites. On the other hand, LPGs and GIPLs were able to activate the production of NO, IL-6, IL-12 and TNF–α preferably via TRL2. However, in CHO cells, only GIPLs were able to activate TRL2 and TRL4. In vivo studies using male guinea pigs (Cavia porcellus) showed that only strain L88 was able to develop more severe ulcerated lesions especially in the presence of salivary gland extract (SGE). CONCLUSION: The two L. enriettii strains exhibited polymorphisms in their LPGs and GIPLs and those features may be related to a more pro-inflammatory profile in the L88 strain.

Leishmania amazonensis is the etiologic agent of the cutaneous and diffuse leishmaniasis often associated with drug resistance. Lapachol [2-hydroxy-3-(3′-methyl-2-butenyl)-1,4-naphthoquinone] displays a wide range of antimicrobial properties against many pathogens. In this study, using the classic microscopic in vitro model, we have analyzed the effects of a series of lapachol and chlorides complexes with antimony (V), bismuth (V), and tin (IV) against L. amazonensis. All seven compounds exhibited antileishmanial activity, but most of the antimony (V) and bismuth (V) complexes were toxic against human HepG2 cells and murine macrophages. The best IC50 values (0.17 ± 0.03 and 0.10 ± 0.11 μg/mL) were observed for Tin (IV) complexes (3) [(Lp)(Ph3Sn)] and (6) (Ph3SnCl2), respectively. Their selective indexes (SIs) were 70.65 and 120.35 for HepG2 cells, respectively. However, while analyzing murine macrophages, the SI decreased. Those compounds were moderately toxic for HepG2 cells and toxic for murine macrophages, still underlying the need of chemical modification in this class of compounds.

Neutrophils are rapidly recruited to the site of Leishmania infection and play an active role in capturing and killing parasites. They are the main source of leukotriene B₄ (LTB₄), a potent proinflammatory lipid mediator. However, the role of LTB₄ in neutrophil infection by Leishmania amazonensis is not clear. In this study, we show that L. amazonensis or its lipophosphoglycan can induce neutrophil activation, degranulation, and LTB₄ production. Using pharmacological inhibitors of leukotriene synthesis, our findings reveal an LTB₄-driven autocrine/paracrine regulatory effect. In particular, neutrophil-derived LTB₄ controls L. amazonensis killing, degranulation, and reactive oxygen species production. In addition, L. amazonensis infection induces an early increase in Toll-like receptor 2 expression, which facilitates parasite internalization. Nuclear factor kappa B (NFkB) pathway activation represents a required upstream event for L. amazonensis-induced LTB₄ synthesis. These leishmanicidal mechanisms mediated by neutrophil-derived LTB₄ act through activation of its receptor, B leukotriene receptor 1 (BLT1).

Several tropical cities are permissive to Aedes aegypti and dengue virus (DENV) endemicity and have allowed for invasion and circulation of Zika virus (ZIKV) in the same areas. People living in arbovirus-endemic regions have been simultaneously infected with ≥2 arboviruses. A. aegypti mosquitoes from Manaus, the capital city of Amazonas State in Brazil, were coinfected with circulating strains of DENV and ZIKV. The coinfected vectors were allowed to bite BALB/c mice. A. aegypti from Manaus is highly permissive to monoinfection and coinfection with DENV and ZIKV and is capable of cotransmitting both pathogens by bite. Coinfection strongly influences vector competence, favoring transmission of ZIKV to the vertebrate host. This finding suggests that A. aegypti is an efficient vector of ZIKV and that ZIKV would be preferentially transmitted by coinfected A. aegypti. Coinfection in the vector population should be considered a new critical epidemiological factor and may represent a major public health challenge.

Chagas disease, caused by the parasite Trypanosoma cruzi , is considered endemic in more than 20 countries but lacks both an approved vaccine and limited treatment for its chronic stage. Chronic infection is most harmful to human health because of long-term parasitic infection of the heart. Here we show that immunization with a virus-like particle vaccine displaying a high density of the immunogenic α-Gal trisaccharide (Qβ-αGal) induced several beneficial effects concerning acute and chronic T . cruzi infection in α1,3-galactosyltransferase knockout mice. Approximately 60% of these animals were protected from initial infection with high parasite loads. Vaccinated animals also produced high anti-αGal IgG antibody titers, improved IFN-γ and IL-12 cytokine production, and controlled parasitemia in the acute phase at 8 days post-infection (dpi) for the Y strain and 22 dpi for the Colombian strain. In the chronic stage of infection (36 and 190 dpi, respectively), all of the vaccinated group survived, showing significantly decreased heart inflammation and clearance of amastigote nests from the heart tissue.

Dengue virus (DENV) and Zika virus (ZIKV) belong to the same viral family, the Flaviviridae. They cause recurring threats to the public health systems of tropical countries such as Brazil. The primary Brazilian vector of both viruses is the mosquito Aedes aegypti . After the mosquito ingests a blood meal from an infected person, the viruses infect and replicate in the midgut, disseminate to secondary tissues and reach the salivary gland (SG), where they are ready to be transmitted to a vertebrate host. It is thought that the intrinsic discrepancies among mosquitoes could affect their ability to deal with viral infections. This study confirms that the DENV and ZIKV infection patterns of nine Ae . aegypti field populations found in geographically separate health districts of an endemic Brazilian city vary. We analyzed the infection rate, disseminated infection, vector competence, and viral load through quantitative PCR. Mosquitoes were challenged using the membrane-feeding assay technique and were tested seven and fourteen days post-infection (early and late infection phases, respectively). The infection responses varied among the Ae . aegypti populations for both flaviviruses in the two infection phases. There was no similarity between DENV and ZIKV vector competencies or viral loads. According to the results of our study, the risk of viral transmission overtime after infection either increases or remains unaltered in ZIKV infected vectors. However, the risk may increase, decrease, or remain unaltered in DENV-infected vectors depending on the mosquito population. For both flaviviruses, the viral load persisted in the body even until the late infection phase. In contrast to DENV, the ZIKV accumulated in the SG over time in all the mosquito populations. These findings are novel and may help direct the development of control strategies to fight dengue and Zika outbreaks in endemic regions, and provide a warning about the importance of understanding mosquito responses to arboviral infections.

Chagas disease, caused by the parasite Trypanosoma cruzi, is considered endemic in more than 20 countries but lacks both an approved vaccine and limited treatment for its chronic stage. Chronic infection is most harmful to human health because of long-term parasitic infection of the heart. Here we show that immunization with a virus-like particle vaccine displaying a high density of the immunogenic [alpha]-Gal trisaccharide (Q[beta]-[alpha]Gal) induced several beneficial effects concerning acute and chronic T. cruzi infection in [alpha]1,3-galactosyltransferase knockout mice. Approximately 60% of these animals were protected from initial infection with high parasite loads. Vaccinated animals also produced high anti-[alpha]Gal IgG antibody titers, improved IFN-[gamma] and IL-12 cytokine production, and controlled parasitemia in the acute phase at 8 days post-infection (dpi) for the Y strain and 22 dpi for the Colombian strain. In the chronic stage of infection (36 and 190 dpi, respectively), all of the vaccinated group survived, showing significantly decreased heart inflammation and clearance of amastigote nests from the heart tissue.

Dengue virus (DENV) and Zika virus (ZIKV) belong to the same viral family, the Flaviviridae. They cause recurring threats to the public health systems of tropical countries such as Brazil. The primary Brazilian vector of both viruses is the mosquito Aedes aegypti. After the mosquito ingests a blood meal from an infected person, the viruses infect and replicate in the midgut, disseminate to secondary tissues and reach the salivary gland (SG), where they are ready to be transmitted to a vertebrate host. It is thought that the intrinsic discrepancies among mosquitoes could affect their ability to deal with viral infections. This study confirms that the DENV and ZIKV infection patterns of nine Ae. aegypti field populations found in geographically separate health districts of an endemic Brazilian city vary. We analyzed the infection rate, disseminated infection, vector competence, and viral load through quantitative PCR. Mosquitoes were challenged using the membrane-feeding assay technique and were tested seven and fourteen days post-infection (early and late infection phases, respectively). The infection responses varied among the Ae. aegypti populations for both flaviviruses in the two infection phases. There was no similarity between DENV and ZIKV vector competencies or viral loads. According to the results of our study, the risk of viral transmission overtime after infection either increases or remains unaltered in ZIKV infected vectors. However, the risk may increase, decrease, or remain unaltered in DENV-infected vectors depending on the mosquito population. For both flaviviruses, the viral load persisted in the body even until the late infection phase. In contrast to DENV, the ZIKV accumulated in the SG over time in all the mosquito populations. These findings are novel and may help direct the development of control strategies to fight dengue and Zika outbreaks in endemic regions, and provide a warning about the importance of understanding mosquito responses to arboviral infections. Author summary Dengue and Zika are neglected diseases caused by viruses transmitted to humans by mosquitoes (vector-borne diseases). The primary vector of both diseases is Aedes aegypti, a highly abundant mosquito in tropical countries and adapted to the urban habitat. The viral cycle in the vector starts when the mosquito bites an infected person and acquires the viruses through the blood meal. When the infected blood reaches the mosquito’s midgut, the viruses invade the epithelial cells and disseminate in several organs until they reach the salivary glands, enabling viral transmission to the next person. However, the mosquitoes have developed strategies to combat the viral invasion and dissemination in their body, making this journey a challenge to the viruses. Herein, we show that the mosquito responses against dengue and Zika viruses are distinct. In addition, mosquitoes from separate populations of the same city have different abilities to deal with the viruses in both cases, dengue and Zika infections. Our results show the diversity of responses that the mosquitoes may present to viral infections. These findings may better direct disease control strategies to combat dengue and Zika outbreaks in endemic regions.